The terms glycan and polysaccharide are defined by IUPAC as synonyms meaning “compounds consisting of a large number of monosaccharides linked glycosidically.” However, in practice the term glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide.
Many secreted eukaryotic proteins possess post-translational carbohydrate modifications of certain asparagine residues (N-glycans). N-glycans become attached to proteins in the endoplasmic reticulum of eukaryotic cells on the nitrogen (N) in the side chain of asparagine in the sequon of a protein. The sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline. N-Glycans are commonly comprised of the sugars galactose, N-acetylneuraminic acid, N-acetylglucosamine (GlcNAc), fucose, and mannose but may also contain other sugars such as N-acetylgalactosamine and N-glycolylneuraminic acid.
Many classes of biologic drugs (e.g., antibodies, fusion proteins, growth factors, cytokines, etc.) are glycoproteins that possess N-glycans. The composition of these glycans can affect the stability, bioactivity, and serum half-life of a biologic drug. As such, glycan structure is designated a critical quality attribute (CQA) that must be monitored during biologic manufacturing, and the glycan profile of a finished product is used to assess the consistency of a manufacturing process from batch to batch.
To assess their composition, glycans can be structurally profiled using a variety of analytical methods including liquid chromatography, mass spectrometry, or capillary electrophoresis. Additionally, enzymes that remove sugars from the non-reducing end of glycans (exoglycosidases) can be used in concert with these analytical methods to sequence glycans and provide an orthogonal assessment of a glycan's structure.
The past decade has seen many advances in the structural analysis of N-glycans. In general, analytical workflows have significantly increased in speed, throughput and sensitivity. This progress has been driven by improvements to nearly every aspect of workflow design including instrument sensitivity, separations technologies, sample preparation and computational analysis of data. Within the past few years, sample preparation for N-glycan profiling has changed dramatically. Improved reagents and methods have simplified and shortened the process of releasing and fluorescently labeling N-glycans. For structural profiling, N-glycans are typically first removed from a peptide or protein using the enzyme PNGase F (New England Biolabs, Ipswich, Mass.). New formats of PNGase F have substantially improved the speed and completeness of N-glycan release from proteins (see for example US 2015/0346194). Following their release, N-glycans are then fluorescently labeled on their reducing end to enable their ultimate detection during downstream analyses. Recently, new chemistries for label attachment have given rise to a new generation of fluorescent labels that offer “instant” labeling of N-glycans and improved sensitivity in downstream analytical methods such as liquid chromatography, mass spectrometry, and capillary electrophoresis (see for example Cohen, et al., Anal Biochem, 211(2):279-287, 1993; US 2012/0107942; and WO 2013/049622).
For decades, exoglycosidases have been used to assist in structural determination of glycans. These enzymes sequentially remove specific terminal sugars from oligosaccharides. Specific and complete removal of the targeted sugar by the exoglycosidase is critical in order to precisely characterize the structure of glycan. Arrays of exoglycosidases with various different specificities can be used to fully identify and order the sugars in N-glycan (and other glycans). The field has typically utilized a series of well-characterized and commercially available exoglycosidases for glycan characterization. These enzymes have performed well in the presence of traditional fluorescent labels that have been attached to N-glycans via Schiff-base chemistry although these reactions can be rather slow. There is, however, a problem with the performance of certain enzymes in the presence of fluorescent labels that have been attached to the glycosylamine at the reducing end of N-glycans via amine reactive chemistry (e.g. reactive carbamate chemistry). For example, one exoglycosidase commonly used in such assays, bovine kidney fucosidase (BKF), does not effectively cleave α(1,6) linked fucose from the core of N-glycans that have been labeled with this class of fluorescent labels. This problem is a major new hurdle for improved methods of enzyme-based structure verification of N-glycans. Another challenge has been performing a defucosylation reaction on a complex N-glycan attached to the glycoprotein or glycopeptide without having to denature the protein or first cleave at least in part, the N-glycan.